Oxygen homeostasis is an essential cellular and systemic function; hypoxia leads to metabolic demise, but this must be balanced by the risk of oxidative damage to cellular lipids, nucleic acids, and proteins resulting from hyperoxia. As a result, cellular and systemic oxygen concentrations are tightly regulated via response pathways that affect the activity and expression of a multitude of cellular proteins. This balance is disrupted in heart disease, cancer, cerebrovascular disease, and chronic obstructive pulmonary disease (Semenza, Genes Dev., 2000, 14, 1983–1991).
The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in homeostatic responses to hypoxia by binding to the DNA sequence 5′-TACGTGCT-3′ and activating the transcription of dozens of genes in vivo under hypoxic conditions (Wang and Semenza, J. Biol. Chem., 1995, 270, 1230–1237). These gene products participate in either increasing oxygen delivery to hypoxic tissues or activating an alternative metabolic pathway (glycolysis) which does not require oxygen. This list includes: aldolase C, enolase 1, glucose transporter 1, glucose transporter 3, glyceraldehyde-3-phosphate dehydrogenase, hexokinase 1, hexokinase 2, insulin-like growth factor-2 (IGF-2), IGF binding protein 1, IGF binding protein 3, lactate dehydrogenase A, phosphoglycerate kinase 1, pyruvate kinase M, p21, transforming growth factor B3, ceruloplasmin, erythropoietin, transferrin, transferrin receptor, a1b-adrenergic receptor, adrenomedullin, endothelin-1, heme oxygenase 1, nitric oxide synthase 2, plasminogen activator inhibitor 1, vascular endothelial growth factor (VEGF), VEGF receptor FTL-1, and p35 (Semenza, Genes Dev., 2000, 14, 1983–1991). Expression of hypoxia-inducible factor-1 alpha is also sensitive to oxygen concentration: increased levels of protein are detected in cells exposed to 1% oxygen and these decay rapidly upon return of the cells to 20% oxygen (Wang et al., Proc. Natl. Acad. Sci. U.S.A., 1995, 92, 5510–5514).
Hypoxia-inducible factor-1 alpha is a heterodimer composed of a 120 kDa alpha subunit complexed with a 91 to 94 kDa beta subunit, both of which contain a basic helix-loop-helix (Wang and Semenza, J. Biol. Chem., 1995, 270, 1230–1237). The gene encoding hypoxia-inducible factor-1 alpha (also called HIF-1 alpha, HIF1A, HIF-1A, HIF1-A, and MOP1) was cloned in 1995 (Wang et al., Proc. Natl. Acad. Sci. U.S.A., 1995, 92, 5510–5514). A nucleic acid sequence encoding hypoxia-inducible factor-1 alpha is disclosed and claimed in U.S. Pat. No. 5,882,914, as are expression vectors expressing the recombinant DNA, and host cells containing said vectors (Semenza, 1999).
Hypoxia-inducible factor-1 alpha expression and HIF-1 transcriptional activity are precisely regulated by cellular oxygen concentration. The beta subunit is a constitutive nuclear protein, while the alpha subunit is the regulatory subunit. Hypoxia-inducible factor-1 alpha mRNA is expressed at low levels in tissue culture cells, but it is markedly induced by hypoxia or ischemia in vivo (Yu et al., J. Clin. Invest., 1999, 103, 691–696). Hypoxia-inducible factor-1 alpha protein is negatively regulated in non-hypoxic cells by ubiquitination and proteasomal degradation (Huang et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 7987–7992). Under hypoxic conditions, the degradation pathway is inhibited, hypoxia-inducible factor-1 alpha protein levels increase dramatically, and the fraction that is ubiquitinated decreases. Hypoxia-inducible factor-1 alpha then translocates to the nucleus and dimerizes with a beta subunit (Sutter et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 4748–4753).
A natural antisense transcript that is complementary to the 3′ untranslated region of hypoxia-inducible factor-1 alpha mRNA has been discovered and is named “aHIF” (Thrash-Bingham and Tartof, J. Natl. Cancer Inst., 1999, 91, 143–151). This is the first case of overexpression of a natural antisense transcript exclusively associated with a specific human malignant disease. aHIF is specifically overexpressed in nonpapillary clear-cell renal carcinoma under both normoxic and hypoxic conditions, but not in papillary renal carcinoma. Although aHIF is not further induced by hypoxia in nonpapillary disease, it can be induced in lymphocytes where there is a concomitant decrease in hypoxia-inducible factor-1 alpha mRNA.
Hypoxia-inducible factor-1 alpha plays an important role in promoting tumor progression and is overexpressed in common human cancers, including breast, colon, lung, and prostate carcinoma. Mutations that inactivate tumor supressor genes or activate oncogenes have, as one of their consequences, upregulation of hypoxia-inducible factor-1 alpha activity, either through an increase in hypoxia-inducible factor-1 alpha protein expression, hypoxia-inducible factor-1 alpha transcriptional activity, or both (Semenza, Pediatr. Res., 2001, 49, 614–617).
Until a tumor establishes a blood supply, the hypoxic conditions limit tumor growth. Subsequent increases in hypoxia-inducible factor-1 alpha activity result in increased expression of target genes such as vascular endothelial growth factor (VEGF). VEGF expression is essential for vascularization and the establishment of angiogenesis in most solid tumors (Iyer et al., Genes Dev., 1998, 12, 149–162). A significant association between hypoxia-inducible factor-1 alpha, VEGF overexpression and tumor grade is also seen in human glioblastoma multiforme, the highest grade glioma in which mean patient survival time is less than one year. The rapidly proliferating tumor outgrows its blood supply, resulting in extensive necrosis, and these regions express high levels of hypoxia-inducible factor-1 alpha protein and VEGF mRNA, suggesting a response of the tumor to hypoxia (Zagzag et al., Cancer, 2000, 88, 2606–2618).
The von Hippel-Landau (VHL) tumor supressor gene product is required for proteolytic destruction of the hypoxia-inducible factor-1 alpha subunit. Hydroxylation of a proline residue on the hypoxia-inducible factor-1 alpha subunit occurs under non-hypoxic conditions, and this is required for recognition by the VHL gene product. Consequently, individuals with errors in the VHL gene have a constitutive hypoxia-like phenotype. Individuals with this disease are predisposed to renal cysts, clear cell renal carcinoma, phaeochromocytoma, haemangioblastomas of the central nervous system, angiomas of the retina, islet cell tumors of the pancreas, and endolymphatic sac tumors (Maxwell et al., Exp. Nephrol., 2001, 9, 235–240). Inactivation of the VHL tumor supressor, coupled with increased levels of hypoxia-inducible factor-1 alpha and VEGF, is responsible for the extensive vascularization of the haemangioblastoma brain tumor.
The p53 tumor supressor also targets hypoxia-inducible factor-1 alpha for degradation by the proteasome. Loss of p53 activity occurs in the majority of human cancers and indicates that amplification of normal hypoxia-inducible factor-1 alpha levels contributes to the angiogenic switch during tumorigenesis (Ravi et al., Genes Dev., 2000, 14, 34–44).
A mouse model of pulmonary hypertension has shown that local inhibition of hypoxia-inducible factor-1 alpha activity in the lung might represent a therapeutic strategy for treating or preventing pulmonary hypertension in at risk individuals. In pulmonary hypertension hypoxia-induced vascular remodeling leads to decreased blood flow, which leads to progressive right heart failure and death. This hypoxia-induced vascular remodeling is markedly impaired in mice that are partially hypoxia-inducible factor-1 alpha deficient (Yu et al., J. Clin. Invest., 1999, 103, 691–696). Decreased vascular density and retarded solid tumor growth is also seen in mouse embryonic stem cells which are deficient for hypoxia-inducible factor-1 alpha (Ryan et al., Embo J, 1998, 17, 3005–3015).
During hypoxia, cells shift to a glycolytic metabolic mode for their energetic needs and hypoxia-inducible factor-1 alpha is known to upregulate the expression of many glycolytic genes. Hypoxia-inducible factor-1 alpha may play a pivotal role in the Warburg effect in tumors, a paradoxical situation in which tumor cells growing under normoxic conditions show elevated glycolytic rates, which enhances tumor growth and expansion. Hypoxia-inducible factor-1 alpha mediates the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3, a gene whose protein product maintains levels of the key regulator of glycolytic flux, fructose-2,6-bisphosphate (Minchenko et al., J. Biol. Chem., 2001, 14, 14).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of hypoxia-inducible factor-1 alpha and to date, investigative strategies aimed at modulating hypoxia-inducible factor-1 alpha function have involved the use of antisense expression vectors and oligonucleotides. These studies have served to define the involvement of hypoxia-inducible factor-1 alpha in disease progression and to identify novel roles of hypoxia-inducible factor-1 alpha in vivo including unique roles for hypoxia-inducible factor-1 alpha as a transcription factor under non-hypoxic conditions and as an inhibitor of gene expression.
Gene transfer of an antisense hypoxia-inducible factor-1 alpha plasmid has been shown to enhance the efficacy of cancer immunotherapy. Antisense therapy was shown to slow, but not eradicate, the growth of EL-4 tumors established in mice. In addition, endogenously expression of hypoxia-inducible factor-1 alpha was almost completely inhibited in these tumors. When antisense therapy was combined with T-cell costimulator B7-1 immunotherapy, the tumors completely and rapidly regressed within 1 week. Furthermore, when these tumor-free mice were rechallenged with EL-4 cells, no tumors emerged, indicating that systemic antitumor immunity had been achieved (Sun et al., Gene Ther., 2001, 8, 638–645).
Activation of hypoxia-inducible factor-1 alpha is thought to aggravate heart failure by upregulation of cardiac ET-1, a gene product involved in heart failure and whose inhibition improves the survival rate of rats with heart failure. In a failing heart, a metabolic switch occurs, and hypoxia-inducible factor-1 alpha activates the expression of glycolytic enzymes as compensation for impaired b-oxidation of fatty acid. Another consequence of increased hypoxia-inducible factor-1 alpha activity is that in rat cardiomyocytes, hypoxia-inducible factor-1 alpha was shown to bind to the 5′-promoter region of the ET-1 gene and increase ET-1 expression. In vitro, an antisense oligonucleotide targeted to hypoxia-inducible factor-1 alpha largely inhibited the increased gene expression of ET-1, confirming the role of hypoxia-inducible factor-1 alpha in heart failure (Kakinuma et al., Circulation, 2001, 103, 2387–2394). This antisense oligonucleotide is comprised of 20 nucleotides and targets bases 11 to 31 of the rat hypoxia-inducible factor-1 alpha with GenBank accession number AF—057308.
Preeclampsia is a disorder of unknown etiology that is the leading cause of fetal and maternal morbidity and mortality. Defective downregulation of hypoxia-inducible factor-1 alpha may play a major role in the pathogenesis of preeclampsia. For most of the first trimester, the human fetus develops under hypoxic conditions but at 10–12 weeks the intervillous space opens, the fetus is exposed to maternal blood and at this stage the trophoblast cells invade the maternal decidua. The switch of the trophoblasts from a proliferative to an invasive phenotype is controlled by cellular oxygen concentration. The proliferative, non-invasive trophoblast phenotype appears to be maintained by hypoxia-inducible factor-1 alpha mediated expression of TGFbeta3 because treatment of human villous explants with an antisense oligonucleotide against hypoxia-inducible factor-1 alpha or TGF beta 3 induces invasion under hypoxic conditions. In this case the hypoxia-inducible factor-1 alpha antisense oligonucleotide was comprised of phosphorothioate oligonucleotides, 16 nucleotides in length, and targeted to the AUG codon (Caniggia et al., J. Clin. Invest., 2000, 105, 577–587.; Caniggia et al., Placenta, 2000, 21 Suppl A, S25–30).
The human intestinal trefoil factor (ITF) gene product protects the epithelial barrier during episodes of intestinal hypoxia. The ITF gene promoter bears a binding site for hypoxia-inducible factor-1 alpha, and the function of hypoxia-inducible factor-1 alpha as a transcription factor for ITF was confirmed in vitro. T84 colonic epithelial cells were treated with a phosphorothioate antisense oligonucleotide, 15 nucleotides in length and targeted to the AUG codon of hypoxia-inducible factor-1 alpha and this resulted in a loss of ITF hypoxia inducibility (Furuta et al., J. Exp. Med., 2001, 193, 1027–1034).
Human epidemiological and animal studies have associated inhalation of nickel dusts with an increased incidence of pulmonary fibrosis. Nickel transcriptionally activates plasminogen activator inhibitor (PAI-1), an inhibitor of fibrinolysis, through the hypoxia-inducible factor-1 alpha signaling pathway. This was evidenced by decreases in PAI-1 mRNA levels when human airway epithelial cells were treated with an antisense oligonucleotide directed against hypoxia-inducible factor-1 alpha identical to the one used in the preeclampsia study discussed above. These data may be critical for understanding the pathology of pulmonary fibrosis and other diseases associated with nickel exposure (Andrew et al., Am J Physiol Lung Cell Mol Physiol, 2001, 281, L607–615).
Hypoxia-inducible factor-1 alpha is constitutively expressed in cerebral neurons under normoxic conditions. A second dimerization partner for hypoxia-inducible factor-1 alpha is ARNT2, a cerebral translocator homologous to hypoxia-inducible factor-1 beta. One splice variant of hypoxia-inducible factor-1 alpha found in rat neurons dimerizes with ARNT2 more avidly than it does with HIF1b, and the resulting hypoxia-inducible factor-1 alpha-ARNT2 heterodimer does not recognize the hypoxia-inducible factor-1 alpha binding site of the erythropoietin gene. This suggests that transcription of a different set of genes is controlled by the hypoxia-inducible factor-1 alpha-ARNT2 heterodimer controls in neurons under nonhypoxic conditions than the hypoxia-inducible factor-1 alpha-hypoxia-inducible factor-1 alpha heterodimer controls under hypoxic conditions. This was evidenced by antisense oligonucleotide downregulation of hypoxia-inducible factor-1 alpha expression in which the antisense oligonucleotide consisted of 16 phosphorothioate nucleotides targeted to bases 38 to 54 of the rat hypoxia-inducible factor-1 with GenBank accession number AF—057308 (Drutel et al., Eur. J. Neurosci., 2000, 12, 3701–3708).
A role for hypoxia-inducible factor-1 alpha in mediating a down-regulatory pathway was recently discovered using antisense oligonucleotide depletion of hypoxia-inducible factor-1 alpha. The peroxisome proliferator-activated receptors (PPARS) are a nuclear hormone-binding proteins that regulate transcriptional activities. Ligands which bind the PPAR-gamma isoform man amplify or inhibit the expression of inflammation-related gene products and may regulate the duration of inflammatory response. Hypoxia elicits a down-regulation of PPAR-gamma in intestinal epithelial cells which is effected through a binding site for hypoxia-inducible factor-1 alpha on the antisense strand of the PPAR-gamma gene. The expression of PPAR-gamma was upregulated in hypoxic cells when treated with an antisense oligonucleotide targeted to hypoxia-inducible factor-1 alpha identical to the one used in the preeclampsia study discussed above (Narravula and Colgan, J. Immunol., 2001, 166, 7543–7548).
As a consequence of hypoxia-inducible factor-1 alpha involvement in many diseases, there remains a long felt need for additional agents capable of effectively inhibiting hypoxia-inducible factor-1 alpha function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of hypoxia-inducible factor-1 alpha expression.
The present invention provides compositions and methods for modulating hypoxia-inducible factor-1 alpha expression.